Objective To understand the respiratory protection competency of staff in hospitals. Methods Staff from six hospitals of different levels and characteristics in Beijing were selected, including doctors, nurses, medical technicians, and servicers, to conduct knowledge assessment on respiratory protection competency. According to exposure risks of respiratory infectious diseases, based on actual cases and daily work scenarios, content of respiratory protection competency assessment was designed from three aspects: identification of respiratory infectious diseases, transmission routes and corresponding protection requirements, as well as correct selection and use of masks. The assessment included 6, 6, and 8 knowledge points respectively, with 20 knowledge points in total, all of which were choice questions. For multiple-choice questions, full marks, partial marks, and no mark were given respectively if all options were correct, partial options were correct and without incorrect options, and partial options were correct but with incorrect options. Difficulty and discrimination analyses on question of each knowledge point was conducted based on classical test theory. Results The respiratory protection competency knowledge assessment for 326 staff members at different risk levels in 6 hospitals showed that concerning the 20 knowledge points, more than 60% participants got full marks for 6 points, while the proportion of full marks for other questions was relatively low. Less than 10% participants got full marks for the following 5 knowledge points: types of airborne diseases, types of droplet-borne diseases, conventional measures for the prevention and control of healthcare-associated infection with respiratory infectious diseases, indications for wearing respirators, and indications for wearing medical protective masks. Among the 20 knowledge questions, 5, 1, and 14 questions were relatively easy, medium, and difficult, respectively; 6, 1, 4, and 9 questions were with discrimination levels of ≥0.4, 0.30-0.39, 0.20-0.29, and ≤0.19, respectively. Conclusion There is still much room for hospital staff to improve their respiratory protection competency, especially in the recognition of diseases with different transmission routes and the indications for wearing different types of masks.